BACKGROUND: Assisted reproductive techniques (ART) are associated with a higher risk of tetralogy of Fallot (TOF) and multiple pregnancies may be associated with a higher risk of congenital anomalies. We assessed the extent to which the association between ART and risk of TOF may be mediated by the higher risk of multiple pregnancies associated with ART. METHODS: We conducted a case-control study using population-based data from the Paris Registry of Congenital Malformations for the period 1987-2009 and a cohort study of congenital heart defects (EPICARD). The study population included 395 cases of TOF and 4104 malformed controls with no known associations with ART. The analysis was based on a path-analysis model using a counterfactual approach, which allows decomposition of the total effect of ART into an indirect effect (that mediated by the association between ART and multiple pregnancies) and a direct effect. RESULTS: ART (all methods combined) were associated with a 2.6-fold higher odds of TOF after adjustment for maternal and paternal characteristics and year of birth (adjusted OR 2.6, 95%CI, 1.5-4.5). Most (79%) of the effect associated with ART was a direct effect (i.e., not mediated by multiple pregnancies), whereas 21% of the effect of ART was due to its association with multiple pregnancies (i.e., the indirect effect). In vitro fertilization with intracytoplasmic sperm injection was associated with a 3.5-fold higher odds of TOF (adjusted OR 3.5, 95%CI, 1.1-11.2); 11% of this effect was mediated through the association of ICSI with multiple pregnancies. CONCLUSIONS: By far, most of the higher risk of TOF associated with ART is a direct effect and only a small proportion of the effect may be mediated by multiple pregnancies.